Mast cells(MCs), basophils, eosinophils, and lymphocytes are cell types integral to the development of an allergic response. Degranulation of MCs and granulocytes, and cytokine production by T cells is induced primarily by cross-linking of the receptor for antigen. However, allergic inflammation may also be generated through activation of receptors coupled to heterotrimeric G proteins (GPCRs). The purpose of this study is to understand mechanisms of intracellular G-protein-coupled signal transduction in immune cells and subsequent pathways to inflammation. GPCRs activate heterotrimeric G proteins, which bind guanosine triphosphate (GTP) in exchange for guanosine diphosphate (GDP). The GTP-bound form of the G protein alpha subunit induces downstream signaling cascades, including intracellular calcium flux responsible for MC/basophil degranulation. This project focuses on a family of regulators of G protein signaling (RGS proteins), which inhibit the function of G alpha-i and G alpha-q, but not G alpha-s, proteins by increasing their GTPase activity. G alpha subunits oscillate between GDP- (inactive) and GTP- (active) bound forms based on ligand occupancy of the associated receptor. The GTPase accelerating (GAP) activity of RGS proteins limits the time of interaction of active G-alpha and its effectors, resulting in desensitization of GCPR signaling. Despite a growing body of knowledge concerning the biochemical mechanisms of RGS action, relatively little is known about the physiological role of these proteins in native mammalian systems. Compounds acting on GPCRs, such as platelet-activating factor (PAF) and adenosine, induce granulocyte and MC degranulation independently of IgE. In previous years'work, we identified an RGS protein, RGS13, which inhibits IgE-mediated mast cell degranulation and anaphylaxis in mice by binding to and counteracting activation of the critical downstream enzyme phosphoinositide-3 kinase (PI3 kinase). We also found that RGS13 regulates GPCR-induced degranulation and cytokine production by human MCs through its GAP activity. These results uncovered a new physiological function of RGS proteins with broad implications for cell growth, metabolism, and immunity: the direct inhibition of PI3 kinase. We hypothesized that abnormalities in RGS13 activity may exist in patients with idiopathic anaphylaxis or other disorders that may be associated with increased mast cell reactivity. Our current focus is to understand regulation of RGS13 expression and function. We found that the tumor suppressor p53 binds to a functional site in the RGS13 promoter, leading to inhibition of RGS13 expression in MCs. During the course of these studies, we also discovered a potential role for p53 in the regulation of MC degranulation and cytokine production. These results suggest that p53 expression and/or function should be examined in clinical disorders of MC degranulation such as anaphylaxis. In the current year's work, we also found that RGS13 was phosphorylated by protein kinase A (PKA). GPCR ligands such as beta-adrenergic agonists generate intracellular cyclic adenosine monophosphate (cAMP), which in turn activates PKA. We mapped the site of RGS13 phosphorylation by PKA and showed that phosphorylated RGS13 protein had a prolonged half-life inside cells. Collectively, these studies have provided insight into the regulation of RGS13 expression, and we have begun to search for polymorphisms affecting RGS13 expression and/or function in patients with anaphylaxis. In contrast to MCs, eosinophils do not express the high affinity receptor for IgE. Thus, mechanisms of eosinophil degranulation are incompletely elucidated. We collaborated with the Eosinophil Biology Section of the LAD to explore mechanisms of GPCR-mediated eosinophil degranulation and cytokine production. PAF is a phospholipid mediator released from activated macrophages, MCs, and basophils that promotes pathophysiologic inflammation. We showed that PAF promoted degranulation (release of eosinophil peroxidase) by a mechanism that was independent of the characterized PAF receptor. Overall, this work provided the first direct evidence of a role for PAF in activating and inducing degranulation of mouse eosinophils, a crucial feature for the interpretation of mouse models of PAF-mediated asthma and anaphylaxis. Likewise, PAF and lysoPAF-mediated activities that are not dependent on signaling through the PAF receptor suggested the existence of other unexplored molecular signaling pathways mediating responses from PAF and closely related phospholipid mediators. The other major area of investigation in this project is the regulation of GPCR-mediated recruitment of inflammatory cells to sites of allergic inflammation. A major class of compounds acting on GPCRs in leukocytes are chemokines. Chemokines and their receptors orchestrate cell trafficking during the immune response. We found that RGS16 was expressed in activated Th1, Th2, and Th17 CD4+ effector T cells. RGS16-deficient T cells migrated more to Th2-associated chemokines such as CCL17 in vitro. These results translated into abnormal T cell trafficking in Th2-associated pathologies. In collaboration with Dr. Thomas Wynn (Laboratory of Parasitic Diseases, NIAID), we showed that Rgs16-/- mice infected with the helminth Schistosoma mansoni had more inflammation, fibrosis, and T cell infiltration in lungs than wild type counterparts. We concluded that RGS16 attenuates Th2 responses to Schistosoma antigens. These results were presented at the American Association of Immunologists meeting in 2010. A second project was to examine the role of RGS16 in experimental autoimmune encephalitis, which is thought to be associated with a Th1-mediated inflammatory response, in collaboration with Dr. Samia Khoury of Harvard Medical School. A preliminary experiment has shown that RGS16-deficient mice have a delayed onset of clinical disease (which resembles the human disease multiple sclerosis) compared to WT mice, but the mechanisms behind this phenotype are still being investigated. We have also embarked on identification of chemokine receptors, G protein, and RGS proteins involved in regulating the function of basophils in allergic inflammation in mice. The cysteine protease papain, which is associated with occupational allergy in humans, was recently shown in published studies to induce antigen-specific Th2 differentiation and antigen-specific IgE production in mice through a basophil-dependent mechanism. Papain immunization of mice leads to accumulation of basophils in draining lymph nodes, and papain activates basophils directly through an unknown sensor and/or surface receptor to produce Th2-inducing cytokines like IL-4. We found increases in chemokine levels in lymph nodes after papain immunization. Basophils expressed several chemokine receptors such as CCR2, CCR4, and CXCL2 and migrated toward a gradient of their cognate ligands in vitro. Basophil recruitment to lymph nodes by papain was strongly diminished in the absence of either CCL17 or the receptors CCR2 or CCR4. These findings have begun to elucidate mechanisms of basophil trafficking in allergic inflammation.